Fowler, P. C. and O'Sullivan, N. C. (2016). ER-shaping proteins are required for ER and mitochondrial network organisation in motor neurons. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27170313Summary:
Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative disorders characterised by degeneration of the longest motor neurons in the corticospinal tract, leading to muscle weakness and spasticity of the lower limbs. Pathogenic variants in genes encoding proteins that shape the endoplasmic-reticulum (ER) network are a leading cause of HSP, however, the mechanisms by which loss of ER-shaping proteins underpin degeneration of selective neurons in HSP remain poorly understood. To begin to address this, a novel in vivo model of HSP in Drosophila melanogaster was generated by targeted knockdown of the ER-shaping protein Arl6IP1. Variants in the human homolog of this gene have recently been linked to HSP subtype SPG61. Arl6IP1 RNAi flies display progressive locomotor deficits without a marked reduction in lifespan, recapitulating key features of HSP in human patients. Loss of Arl6IP1 leads to fragmentation of the smooth ER and disrupted mitochondrial network organisation within the distal ends of long motor neurons. Furthermore, genetically increasing mitochondrial fission, by overexpression of Dynamin related protein 1 (Drp1), restores mitochondrial network organisation and rescues locomotor deficits in two independent Drosophila models of HSP. Taken together, these results propose a role for ER-shaping proteins in mitochondrial network organisation in vivo and suggest that impaired mitochondrial organisation may be a common mechanism underpinning some forms of HSP.

Rana, A., Oliveira, M. P., Khamoui, A. V., Aparicio, R., Rera, M., Rossiter, H. B. and Walker, D. W. (2017). Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster. Nat Commun 8(1): 448. PubMed ID: 28878259Summary:
The accumulation of dysfunctional mitochondria has been implicated in aging, but a deeper understanding of mitochondrial dynamics and mitophagy during aging is missing. This study shows that upregulating Drp1-a Dynamin-related protein that promotes mitochondrial fission-in midlife, prolongs Drosophila lifespan and healthspan. Short-term induction of Drp1, in midlife, is sufficient to improve organismal health and prolong lifespan, and observe a midlife shift toward a more elongated mitochondrial morphology, which is linked to the accumulation of dysfunctional mitochondria in aged flight muscle. Promoting Drp1-mediated mitochondrial fission, in midlife, facilitates mitophagy and improves both mitochondrial respiratory function and proteostasis in aged flies. Finally, autophagy is required for the anti-aging effects of midlife Drp1-mediated mitochondrial fission. These findings indicate that interventions that promote mitochondrial fission could delay the onset of pathology and mortality in mammals when applied in midlife. Mitochondrial fission and fusion are important mechanisms to maintain mitochondrial function. Here, the authors report that middle-aged flies have more elongated, or 'hyper-fused' mitochondria, and show that induction of mitochondrial fission in midlife, but not in early life, extends the health and life of flies.

Ordonez, D. G., Lee, M. K. and Feany, M. B. (2018). alpha-synuclein induces mitochondrial dysfunction through spectrin and the actin cytoskeleton. Neuron 97(1): 108-124.e106. PubMed ID: 29249285Summary:
Genetics and neuropathology strongly link alpha-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related alpha-synucleinopathies. This study describes a new Drosophila model of alpha-synucleinopathy based on widespread expression of wild-type human alpha-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant alpha-synuclein aggregation. Results of forward genetic screening and genetic analysis were used in this new model to demonstrate that alpha-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse alpha-synucleinopathy model and in postmortem brain tissue from patients with alpha-synucleinopathy. Importantly, evidence is provided that the interaction of alpha-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for alpha-synuclein induced neurodegeneration.

Mitochondria in drp-12 and 3665/13510hemocytes were extensively tubular when compared with wild-type mitochondria. Quantifying mitochondrial morphology revealed a 2-fold decrease in the number of mitochondria and a significant increase in the contribution of tubular and extensively tubular mitochondria to the total mitochondrial CSA in drp-1 mutant hemocytes when compared with wild-type cells. Interestingly, 13510/+ hemocytes or eye disc cells displayed a dominant mitochondrial fission defect that was completely rescued by a genomic duplication of drp-1. The mitochondrial fission defect in mutant cells could result from a reduced mitochondrial association of Drp-1 (Goyal, 2007).

An increase in mitochondrial contiguity due to a loss of Drp-1 function was also confirmed by measuring fluorescence recovery after photobleaching (FRAP) of Mito-YFP in drp-1 RNAi-treated S2R+ cells that had extensively tubular mitochondria. Relative FRAP of Mito-YFP in a defined mitochondrial region in drp-1 RNAi-treated cells was significantly higher than that observed in mock RNAi-treated cells (Goyal, 2007).

drp-1 mutant hemocytes were protected from etoposide-induced death up to at least 10 hr, as revealed by a lack of caspase activation, PS exposure, or PI permeability in the majority (~80%) of these cells. Furthermore, drp-1 mutant and dsRNA-treated S2R+ cells were significantly protected from cycloheximide-, actinomycin-D-, or UV-B-induced death. Consistent with increased protection, mitochondria in the majority (~98%) of etoposide-treated drp-12 hemocytes failed to fragment. Interestingly, mitochondria in etoposide-treated 3665/13510 hemocytes revealed a tubular, yet beaded and swollen intermediate in mitochondrial fragmentation by 4 hr that yielded some fragmented mitochondria in few (~25%) cells later. Therefore, reduced (drp-12) or delayed (3665/13510) mitochondrial fragmentation decreased effector caspase activation and protected cells from genotoxic stress. Moreover, an increase in expression of Drp-1 in hemocytes resulted in enhancement of etoposide-induced cell death (Goyal, 2007).

The majority (~70%) of the C6-ceramide-treated drp-12 hemocytes did not show effector caspase activation or PS exposure and displayed significant protection, similar to what was observed with etoposide, although hemocytes derived from the weaker allelic combination, 13510/3665, were apoptotic. Unlike 13510/3665 mitochondria, drp-12 mitochondria failed to fragment, consistent with an essential role for Drp-1-mediated mitochondrial fragmentation during apoptosis in Drosophila. Moreover, developmental PCD in drp-12 mutant larvae was considerably reduced, as revealed by the enlarged central nervous system and a prominently elongated ventral ganglion, similar to other PCD-defective mutants reported (Mills, 2006; Goyal, 2007).

During metamorphosis, the first ecdysone pulse triggers mitochondrial fragmentation in prepupal tissues, although it is after the second ecdysone pulse that salivary gland histolysis occurs. It is likely that DIAP-1 inhibits caspases in these cells that have fragmented mitochondria until it is downregulated at the transcriptional level or degraded after the second ecdysone pulse (Yin, 2005). Interestingly, this was mimicked ex vivo in etoposide-treated DIAP-1+ hemocytes (Goyal, 2007).

The data presented in this study show involvement of mitochondrial fragmentation for ARK-mediated Dronc activation during cell death. The RHG-family proteins that localize to the mitochondria (Claveria, 2002; Haining, 1999; Olson, 2003) might activate Drp-1-mediated mitochondrial fragmentation. This could result in exposure of cytochrome-C (Varkey, 1999) or release of Peanut (Gottfried, 2004), which antagonize DIAP-1-mediated suppression of Dronc. However, since Drosophila PCD is unaffected upon knockdown of cytochrome-C (Dorstyn, 2004), mitochondrial fragmentation in Drosophila and mammalian cells would increase mitochondrial surface area and perhaps the concentration of bulky head group lipids on the outer mitochondrial membrane, facilitating recruitment of proapoptotic proteins. Drp-1 might organize sites for Drosophila Bcl-2-family protein Debcl function on mitochondria (Dorstyn, 2002) that are similar to mitochondrial sites of Bax recruitment in mammalian cells (Karbowski, 2002; Goyal, 2007 and references therein).

These results provide the first evidence that Drp-1-mediated mitochondrial fragmentation upstream of effector caspase activation modifies apoptotic sensitivity. Thus, mitochondrial fragmentation, like caspase activation, plays a conserved and unifying role in diverse cell death pathways from worms to mammals (Frank, 2001; Jagasia, 2005). Although the function of the highly contiguous mitochondria during lipid-induced cell death remains poorly understood, this study brings to the forefront a modulatory role for mitochondrial remodeling in determining the susceptibility of Drosophila cells to death (Goyal, 2007).

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, the Drosophila Pink1knockout (KO) model was used. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also
display a reduced mitochondrial ATP synthesis. Because mitochondrial
dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, whether fission machinery deficits underlie the bioenergetic defect in Pink1- KO flies was investigated. It was found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity
in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely
derived from a defect in the ETC complex assembly, which can be
partially rescued by increasing mitochondrial fission. Taken together,
these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics (Liu, 2011).

Ample evidence indicates that mitochondrial dysfunction plays
a pivotal role in the development of Parkinson's disease (PD). A 30%-40% reduction of mitochondrial electron transport chain (ETC) complex I activity has been observed in the postmortem brains of idiopathic PD patients. 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) and rotenone, inhibitors of ETC
complex I, induce clinical and pathological manifestations that
recapitulate cardinal PD symptoms in humans and in animal
models, supporting the hypothesis that mitochondrial
bioenergetic defects contribute to PD pathogenesis (Liu, 2011).

The significance of mitochondrial dysfunction in the development
of PD was further strengthened by the discovery of
PINK1 as the causal gene of PARK6. PINK1 encodes a mitochondrial
kinase, but its physiological role remains to be elucidated.
Reduction or loss of PINK1 causes bioenergetic deficits
that include loss of membrane potential, calcium buffering, ATP
synthesis rate, and respiration in cell culture systems. In Drosophila and mouse PINK1-KOs, decreased ETC complex I mediated respiration and ATP content have been reported. ETC complex assembly depends on inner mitochondrial membrane integrity, which is maintained by fusion and fission processes. Fusion and fission regulate the number, size, and morphology of mitochondria in a dynamic manner, and perturbing
these processes could affect membrane stability. Several key
molecules that regulate these delicate processes have been identified:
Dynamin-like GTPase (Drp1) is a key molecule in fission, whereas mitofusin (Mfn) and optic atrophy 1 (Opa1) play a major role in fusion. Mutations in Mfn 2 and Opa1 result in Charcot-Marie-Tooth neuropathy and autosomal dominant
optical atrophy, respectively (Liu, 2011 and references therein).

Pink1-KO flies have deficits in mitochondrial fission and abnormal
mitochondrial morphology, which can be alleviated by
one additional copy of Drp1 (Yang, 2008; Poole, 2008; Deng; 2008), indicating that the fission machinery contributes to the mitochondrial pathology in flies.
This study reports that the fission machinery defect contributes to
bioenergetic deficits in the Pink1-KO flies by impairing ETC
complex assembly. This finding raised the possibility that these
biochemical and mitochondrial dysfunctions in Pink1 flies are
also the basis of human PD patient pathogenesis. It is therefore
important to validate these in human patients (Liu, 2011).

Loss of Pink1 leads to impaired ETC
complex IV function, and previous observations were confirmed that Pink1-KOs also have ETC complex I defects. These data provide strong support to the notion that mitochondrial OXPHOS defects are integral parts of PD pathogenesis.
It is important to note that deficits in ETC complex I were
reported in human PD patients, as well as in pharmacological
mouse models of PD, and deficits in ETC complex IV
were found in an α-synuclein transgenic mouse model of PD.
Most importantly, the current findings reveal that defective OXPHOS
complex assembly underlies the impaired OXPHOS function. It
will be interesting to investigate whether this mechanism is also
true in human patients with PINK1 mutations (Liu, 2011).

This study further revealed that OXPHOS complex assembly
and function is modulated by mitochondrial dynamics and involves
the fission machinery. In this case, Drp1 partially rescued the deficits
of OXPHOS assembly and function. It is possible that Drp1
promotes mitochondrial fission, which contributes to 'regenerating'
functional competent mitochondria through redistribution of
mitochondrial DNA, RNA, and proteins. OXPHOS complex
assembly could be improved by a 'rejuvenated' population of mitochondria
in the Pink1-KO/Drp1. Alternatively, Drp1 could achieve
its beneficial effect by promoting the clearance of damaged
mitochondria through processes such as autophagy and mitophagy.
This possibility is supported by recent evidence that Pink1 was
implicated in the clearance of damaged mitochondria. It will be interesting to investigate how PINK1 affects mitophagy in vivo, and how mitochondrial dynamics influences mitophagy. Based on the current data, a working hypothesis is proposed involving a bifurcated pathway for PINK1 pathogenesis as follows: the
direct path may involve reduced or lack of phosphorylation of
OXPHOS complex subunits by PINK1, which impairs OXHOS
complex assembly and function. The indirect path may involve
Drp1 and other intermediates that are important for mitochondrial
membrane stability and dynamics, thereby affecting
OXPHOS complex assembly and function. With the assays and
system this study established, these pathways can now be dissected in greater molecular details, especially with the power of Drosophila genetics. Furthermore, if these findings are validated in human PD patients, the knowledge of this aspect of pathogenesis is important for designing therapeutic strategies (Liu, 2011).

Signaling by Folded gastrulation is modulated by mitochondrial fusion and fission

Mitochondria are increasingly being identified as integrators and regulators of cell signaling pathways. Folded gastrulation (Fog) is a secreted signaling molecule best known for its role in regulating cell shape change at the ventral furrow (VF) during gastrulation in Drosophila. Fog is thought to signal via a G-protein coupled receptor, to effect downstream cytoskeletal changes necessary for cell shape change. However, the mechanisms regulating Fog signaling that lead to change in cell morphology are poorly understood. This study describes identification of proteins involved in mitochondrial fusion and fission as regulators of Fog signaling. Pro-fission factors were found to function as enhancers of signaling, while pro-fusion factors were found to have the opposite effect. Consistent with this, activation of Fog signaling was seen to result in mitochondrial fragmentation and inhibiting this process could attenuate Fog signaling. The findings here show that mitochondria, through regulation of fusion-fission, function as downstream effectors and modulators of Fog signaling and Fog dependent cell shape change (Ratnaparkhi, 2013).

Fog is a secreted signaling molecule known to regulate apical constriction of cells at
the ventral furrow during gastrulation in Drosophila. This study describes a genetic
screen conducted to identify regulators of Fog signaling using the adult wing as an
assay system. Fog is expressed in the wing disc; heterozygous mutant combinations of
fog, concertina and dRhoGEF2, result in deformed wings.
Given a functional role for Fog in wing development, it seemed relevant to screen for
interactors using the wing as an assay system. Through the identification of proteins
involved in mitochondrial fusion and fission as regulators of Fog signaling, this study shows,
for the first time, a role for the mitochondria as a downstream effectors of Fog
signaling (Ratnaparkhi, 2013).

Overexpression of Fog leads to mitochondrial fission. A similar phenotype was
observed upon expression of a constitutively active form of concertina suggesting that
this effect is a consequence of the signaling pathway and not Fog protein per se. Conversely, knockdown of Fog results in different mitochondrial morphologies all
of which are associated with excessive or enhanced fusion. In the present study,
expression of fog dsRNA lead to formation of highly filamentous, 'donuts' shaped or
fused amorphous forms of mitochondria.
Whether these different morphologies arise due to differences in the extent of fog
knockdown is not clear at this point (Ratnaparkhi, 2013).

Fog mediated mitochondrial fission is dependent on Drp1 (Dynamin related protein 1).
Inhibiting fission through
down regulation of drp1 suppresses Fog signaling. Consistent with this,
knock down of drp1 in cells that respond to Fog in the blastoderm, leads to
invagination defects at the ventral furrow. Interestingly drp1 has been identifed as a differentially expressed factor at the ventral
furrow. Injection of dsRNA against drp1 was found result in ventral furrow defects
ranging from mild to severe. In the experiments described here,
the ventral furrow phenotype due to transgenic knockdown of drp1 were not as severe
as those observed in fog mutants; one reason for this could be insufficient expression
of drp1 dsRNA. Relatively stronger effects were seen in embryos in which expression
of the RNAi was carried out at 28°. Some of these phenotypes were also
observed in drp1KG03815 mutants. However, the posterior
midgut primordium (PMG) phenotype
associated with fog loss-of-function was not seen in these mutants. One reason for this
could be the presence of maternal drp1 mRNA that compensates for the loss of any
zygotic drp1 expression. Nonetheless, the observation that inhibiting mitochondrial
fission results in fog loss-of –function like phenotypes correlates well with observed
interaction between fog and drp1 and suggests that mitochondrial fission is necessary
for coordinated cell shape change at the ventral furrow (Ratnaparkhi, 2013).

The differential effect of fog gain-of-function and loss-of-function on mitochondrial
morphology also indicates that the system might be used by the cell to 'sense' and
distinguish one scenario from the other, pointing towards a very sensitive role for
mitochondria in regulating Fog signaling. In this context, it would be interesting to
test if the production of reactive oxygen species (ROS) or any other 'mitochondrial
output' is altered in response to Fog signaling, since this could be an additional
parameter the cell could use, to respond appropriately to Fog. It would also be
interesting to see if the involvement of mitochondria in regulation of cell shape
change is more widespread that previously known. This will need to be tested more
rigorously. It would also be interesting to determine if the effect of fog on
mitochondrial morphology is context dependent (Ratnaparkhi, 2013).

Generation of cell asymmetry and its regulation by mitochondrial fusion and fission
pathways has been previous observed in migrating lymphocytes. In these cells,
activation of G-protein signaling in response to chemokines was found to result in the
redistribution and accumulation of mitochondria in the uropod or non migrating front
to provide the ATP essential for actomyosin contraction during migration. The
process of redistribution was shown to dependent on mitochondrial fission such that
inhibiting fission lead to a loss of cell polarization and inhibition of migration. More recently, in another study, mitochondrial fission mediated by drp1 was found to be necessary and
sufficient for delamination of cells of the amnioserosa (AS) during dorsal closure- a
process that occurs via acto-myosin contraction. While these studies together with the
one described in this study suggest a wider role for mitochondria in regulating acto-myosin
based contraction and cell shape change, fragmentation of mitochondria in response
to Fog may be required for relocalization of mitochondria to the site of action to
provide the necessary energy for apical constriction (Ratnaparkhi, 2013).

How might Fog regulate mitochondrial fragmentation? One possibility is that
activation of Fog signaling modulates expression or localization of drp1 in a manner
that promotes mitochondrial fission. Drp1 is largely a cytoplasmic protein, which gets
recruited to the mitochondria during fission. Many studies on mammalian drp1 have
shown the recruitment to be regulated by post-translational modifications such as
phosphorylation and sumoylation. However, based on the
interaction between Fog and actin regulators it is likely that actin may be involved in
mediating fission. Recent studies have shown that actin can regulate recruitment of
Drp1 to mitochondria in a context dependent and thus control the process of fission. In another study excessive stable F-actin was shown to accumulate Drp1 and prevent it from localizing
to mitochondria in a process dependent on the non-muscle Myosin II resulting in long
elongated mitochondria. The suppression of Fog mediated
mitochondrial fission by expression of WASP, an actin nucleator, suggests that a
similar mechanism could be operating in this context as well (Ratnaparkhi, 2013).

This study was initiated by identification of rhomboid-7 as a suppressor of fog. At this
point, it is unclear how Rho-7, a pro-fusion factor might interact with Fog signaling.
It is likely that the interaction is independent of its role in mitochondrial fusion. This
will need further investigation (Ratnaparkhi, 2013).

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. This paper demonstrates a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1–depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis (Mitra, 2012).

The Drosophila follicle cell layer encapsulates egg chambers containing 15 nurse cells and one oocyte. The follicle cells comprising this cell layer progress through different developmental stages. During stages 1-5 (S1-5), most follicle cells undergo mitotic divisions, with a few cells exiting the mitotic cycle under Notch activation to form stalk cells separating consecutive egg chambers. During S6-8, all follicle cells exit the mitotic cycle in response to Notch activation and differentiate into an endocycling, polarized epithelium patterned into posterior follicle cells (PFCs), main body cells (MBCs), and anterior follicle cells (AFCs). To examine the effect of inhibiting mitochondrial fission activity in this system, Drosophila follicle cell clones where generated mozygous for a functionally null allele of DRP1 called drp1KG. Clones were identified by lack of a ubiquitin promoter-GFP (UbiGFP) label in their nucleus. The potentiometric dye tetramethylrhodamine ethyl ester (TMRE), which incorporates into the mitochondrial matrix, was used to label mitochondria (Mitra, 2012).

In an S10 egg chamber, nonclonal cells containing a nuclear UbiGFP label have mitochondrial elements widely distributed. Microirradiation at a single point within mitochondria of these cells triggers depolarization (i.e., loss of fluroescent TMRE signal) only at the irradiated site, with little loss of TMRE outside the microirradiated site. This suggested the mitochondrial network of these cells is discontinuous. In drp1KG clones (no UbiGFP label), mitochondria were tightly clustered in a small region of each cell. Single-point microirradiation of mitochondria in a drp1KG clone depolarizes the cell's entire mitochondrial cluster, with complete loss of TMRE signal in 5 s. This indicated that mitochondria in drp1KG clones are highly fused. Reduced mitochondrial fission in drp1KG clones, therefore, causes normally fragmented mitochondrial elements in follicle cells to hyperfuse into a tight cluster (Mitra, 2012).

Next, weather presence of drp1KG clones affects follicle epithelial layer organization was examined. In S6-8 egg chambers, follicle cells normally form a single epithelial monolayer. The presence of drp1KG clones, however, disrupts this monolayer arrangement. The effect is most striking in the PFC region, in which drp1KG clones massively overproliferate. The overpopulated clones undergo mitotic cycling even at S10 or later: they incorporate BrdU, demonstrating that they synthesize DNA, and stain with pH3 antibody, indicating that they transit through mitosis. Surrounding heterozygous tissue and drp1KG MBC clones, in contrast, are postmitotic: they neither incorporate BrdU nor stain for pH3. DRP1 depletion thus prevents cell cycle exit primarily in drp1KG PFC clones, leading to their overpopulation in postmitotic egg chambers (Mitra, 2012).

Hnt expression is rescued in all drp1KG PFC clones generated in the background of HA-DRP1 and in 43% of drp1KG PFC clones with DRP1 reintroduced into them. In both conditions, DRP1 expression prevented the clustered mitochondrial phenotype. Lack of differentiation in drp1KG PFC clones, therefore, results from loss of DRP1 activity (Mitra, 2012).

Down-regulation of Marf-1, the Drosophila homologue of mitofusins (Deng, 2008), combined with DRP1 down-regulation in drp1KG PFC clones causes 22% of the clones to now partially express Hnt. Because Marf-1 RNAi expression causes mitochondrial fragmentation when expressed alone or in drp1KG PFC clones, it was concluded that fragmentation of mitochondria reverses the differentiation block in drp1KG PFCs. Therefore, DRP1-driven mitochondrial fission is required for PFCs to differentiate. Loss of function of the inner mitochondrial membrane fusion protein OPA1 caused cell death in this system (Mitra, 2012).

Differentiation of Drosophila follicle cells requires Notch receptor activation. Upon ligand binding, the Notch receptor is cleaved to release the Notch intracellular domain (NICD), which redistributes into the nucleus to activate genes required for differentiation. To investigate whether DRP1-driven mitochondrial fission activity acts upstream or downstream of Notch activation in driving PFC differentiation, whether NICD is cleaved and released from the plasma membrane was examined in drp1KG PFC clones. Significant NICD levels are retained on the plasma membrane in drp1KG PFC clones marked by CD8GFP relative to nonclonal cells in S6-8 egg chambers. The Notch extracellular domain (NECD) is also retained on the plasma membrane in these clones, confirming that Notch is inactive. In addition, Cut down-regulation, which occurs in response to Notch activation, does not occur in drp1KG PFC clones. DRP1-driven mitochondrial fission activity thus acts upstream of Notch activation to drive PFC differentiation (Mitra, 2012).

NICD loss from the membrane (indicative of Notch activation) increases by 28.2% in drp1KG PFC clones after Marf-1 down-regulation. This suggested that Notch inactivation in drp1KG PFC clones is related to mitochondria being highly fused, with mitochondrial fission a prerequisite for Notch receptor activation in the PFCs. Importantly, expression of an activated Notch (N-Act) domain in drp1KG PFC clones partially overrides the differentiation block in 53% of drp1KG PFC clones, resulting in Hnt expression in these clones. As this occurs without the fused mitochondrial morphology of drp1KG PFC clones changing, the data confirmed that DRP1's role in triggering PFC differentiation is upstream of Notch (Mitra, 2012).

Why is DRP1's role in triggering follicle cell differentiation specific to PFCs? Indeed, drp1KG MBC clones show no differentiation block, as Notch activation still occurs in drp1KG MBC clones. Higher levels of bound DRP1 was found in PFCs compared with MBCs after cell permeabilization with digitonin, which may reflect different mitochondrial morphology between PFCs and MBCs. Supporting this, in S6-8 ovarioles it was found that mitochondria in PFCs exist as dispersed fragments both apically and basolaterally, whereas mitochondria in MBCs are tightly clustered at the lateral side of the nucleus. After S9, no observable differences were seen in mitochondrial morphology (Mitra, 2012).

Fluorescence loss in photobleaching (FLIP) experiments in follicle cells of S6-8 egg chambers revealed that the dispersed mitochondria of PFCs have less matrix continuity relative to the fused mitochondrial cluster of MBCs. Furthermore, single-point microirradiation caused a 44% loss in TMRE mitochondrial signal per MBC compared with a 12% loss per PFC. The rapid loss of mitochondrial TMRE signal in MBCs was similar to drp1KG clonal cells, with mitochondrial morphology in wild-type MBCs indistinguishable from that of drp1KG MBC clones. Together, the observed differences in mitochondrial organization and bound DRP1 levels in PFCs and MBCs suggested greater DRP1-driven mitochondrial fission activity occurs in PFCs relative to MBCs. This corroborates findings that PFCs, unlike MBCs, differentiate under the influence of DRP1 (Mitra, 2012).

PFCs are known to be specified by EGF receptor (EGFR) signaling. In egfrt1/egfrt1 egg chambers (hypomorphic allele of EGFR), mitochondria in PFCs are primarily clustered to one side of the nucleus, in contrast to those in wild-type or egfrt1/+ egg chambers, in which mitochondria are dispersed throughout cells. A similar clustering of mitochondria occurs when a dominant-negative (DN) form of EGFR (EGFR-DN) is clonally expressed in the PFC population. Because PFC mitochondria cluster/fuse in the absence of EGFR signaling, the data suggest that EGFR activation in PFCs promotes mitochondria fragmentation in these cells. This could explain why MBCs, which do not receive the EGFR signal, have fused mitochondria. The underlying basis for how EGFR signaling influences mitochondrial dynamics (by altering fission or fusion components) requires further investigation (Mitra, 2012).

Interestingly, PFCs expressing EGFR-DN did not escape differentiation in spite of having clustered mitochondria. This may imply that a highly fused mitochondrial cluster may only allow escape from differentiation in the context of activated EGFR signaling. Indeed, EGFR-DN expression in drp1KG PFC clones (with fused mitochondria) partially induces differentiation (i.e., Hnt expression) in 40% of the clonal cells compared with no Hnt expression in drp1KG PFC clone. Expression of an activated form of EGFR (EGFR-Act) did not induce differentiation in drp1KG PFC clones. This explains why MBCs, which are not exposed to the EGFR ligand, do not proliferate under DRP1 down-regulation. Thus, cross talk exists between mitochondria and the EGFR signaling pathway in postmitotic PFCs, which helps cells decide whether to differentiate or continue in the mitotic cycle (Mitra, 2012).

Because of DRP1's role in differentiation, lack of DRP1 should generate developmental defects. Consistently, DRP1 down-regulation in early follicle cells in the germarium inhibits stalk cell formation, required to separate consecutive egg chambers. The missing stalk cells in egg chambers, encapsulated by early drp1KG follicle cell clones, leads to fused egg chambers containing pH3-labeled drp1KG clonal cells that lack UbiGFP. FasIII-enriched polar cells, known to induce stalk cells, are seen in wild-type ovarioles but are absent in the drp1KG clonal follicle cell population. Lack of polar cells is not the basis of cell proliferation of drp1KG PFCs because FasIII-positive polar cells appear in the surrounding heterozygous tissue. In addition, compound egg chambers with drp1KG follicle stem cell clones frequently arise, including egg chambers with 30 nurse cells and two oocytes (Mitra, 2012).

Down-regulation of DRP1 also causes developmental defects in the postmitotic follicle cell layer. There, in 22% of the cases, drp1KG PFC clones fail to trigger migration of the oocyte nucleus toward the anterior. The postmitotic stage drp1KG phenotypes resemble loss of function of the Hippo-Salvador-Warts pathway, which has tumor suppressor effects in higher organisms, including mice (Mitra, 2012).

The observed link between cell differentiation and mitochondrial fission state during oogenesis could relate to cyclin E, which controls S-phase entry. Indeed, inhibition of mitochondrial ATP synthesis in a cytochrome oxidase mutant promotes specific degradation of cyclin E (but not other cyclins) and blocks S-phase entry in Drosophila. In fibroblasts, cyclin E levels increase under conditions of DRP1 inhibition. In Drosophila follicle cells cyclin E levels were found to increase when DRP1 is down-regulated and decrease when Marf-1 is down-regulated. This suggests that DRP1-driven mitochondrial fission activity may cause cell cycle exit by lowering cyclin E levels to allow differentiation (Mitra, 2012).

The results support a model in which mitochondrial fission/fusion dynamics regulates cell differentiation across the follicle cell layer of the Drosophila ovariole (see A model for mitochondria's role in cell fate determination). In mitotic stages, increased DRP1-driven mitochondrial fission is required for cell cycle exit as noted in premature DRP1-dependent differentiation of Marf-1 RNAi clones and enhanced proliferation of drp1KG clones. During postmitotic transition, activation of EGFR in the posterior region causes mitochondrial fragmentation. This, in turn, permits cell cycle exit and Notch activation, which drives PFC differentiation. In drp1KG PFC clones with fused mitochondria, therefore, Notch remains inactive, and cells proliferate. In the main body region, not exposed to the EGFR ligand, postmitotic differentiation and patterning occur in the absence of DRP1. Thus, cell proliferation/differentiation mechanisms have an intimate relationship to mitochondrial morphology and function during follicle layer development (Mitra, 2012).

Mitochondria shape is controlled by membrane fusion and fission mediated by mitofusins, Opa1, and Drp1, whereas mitochondrial motility relies on microtubule motors. These processes govern mitochondria subcellular distribution, whose defects are emphasized in neurons because of their polarized structure. This study examined how perturbation of the fusion/fission balance affects mitochondria distribution in Drosophila axons. Knockdown of Marf or Opa1 resulted in progressive loss of distal mitochondria and in a distinct oxidative phosphorylation and membrane potential deficit. Downregulation of Drp1 rescued the lethality and bioenergetic defect caused by neuronal Marf RNAi, but induced only a modest restoration of axonal mitochondria distribution. Surprisingly, Drp1 knockdown rescued fragmentation and fully restored aberrant distribution of axonal mitochondria produced by Opa1 RNAi; however, Drp1 knockdown did not improve viability or mitochondria function. These data show that proper morphology is critical for proper axonal mitochondria distribution independent of bioenergetic efficiency. The health of neurons largely depends on mitochondria function, but does not depend on shape or distribution (Trevisan, 2018).

Mitochondria are organelles present in all eukaryotic cells and preside over a wide variety of crucial functions, including respiration and energy production. Mitochondria are organized in a fluidly interconnected, dynamic network, and their structure and position are not fixed but vary by cell type, developmental stage, and physiological context. The dynamic behavior of the network is important for mitochondria distribution and inheritance; is important for remodeling during development and coordination of cell death programs; and allows cells to respond to shifting needs at different intracellular locations (Trevisan, 2018).

Mitochondrial fusion and fission control the shape of mitochondria. The molecular machines that mediate mitochondrial division and fusion are dynamin-related proteins. Drp1 drives the scission of mitochondrial membranes and Mfn1/2 and Opa1 mediate fusion of the outer and inner mitochondrial membranes, respectively (Westermann, 2008). The motility of mitochondria within the cell depends on the action of microtubule-based motors. An ensemble of fission, fusion, and motility governs the distribution of mitochondria within the cell. This is especially evident in neurons whose polarized structure, which is characterized by the presence of axons and dendrites, emphasizes defects in the distribution of mitochondria. A growing body of evidence suggests that fusion and fission affect spatial distribution of mitochondria in neurons. Loss of Drp1, Marf/MFN, or Opa1 in Drosophila depletes mitochondria from neuromuscular junction synapses and motor axons. Mutations of the mitochondrial outer membrane fusion protein Mfn2 or the inner membrane fusion protein Opa1 change the distribution of axonal mitochondria, and knockdown of Opa1 causes redistribution of dendritic mitochondria. It also has been proposed that a direct interaction exists between Mfn2 and the molecular machinery of mitochondrial transport (Trevisan, 2018).

In addition to shape and distribution, the impact of alterations in the activity of mitochondrial dynamics proteins also resonates in mitochondrial function. Substantial evidence has been obtained in mammalian cells, indicating that loss of Mfn2 and Mfn1 function causes alterations in mitochondrial metabolism with loss of mitochondrial membrane potential and reduced endogenous respiration. Furthermore, Opa1 knockdown causes a widespread loss of mitochondrial membrane potential and a reduction in basal respiration. The general tenet is that interconnected mitochondrial networks are found in respiratory active cells, whereas small and fragmented mitochondria are more prevalent in respiratory inactive cells (Trevisan, 2018).

A number of biologically relevant questions thus arise. How are morphology and function linked to the subcellular distribution of mitochondria? Since it is thought that metabolically dysfunctional mitochondria are transported to the neuron body for clearance, is decreased respiration and the consequent retrograde transport of mitochondria triggered by fusion/fission defects responsible for aberrant distribution of axonal mitochondria? Is abnormal mitochondria morphology resulting from fusion/fission defects the reason for this aberrant distribution? Are morphology and function always linked? Do they together contribute to abnormal mitochondria transport? These questions carry also key medical relevance as mutations in Mfn2, Opa1, and Drp1 cause hereditary neuropathies whose pathophysiologic mechanism has been proposed to be abnormal transport and distribution of mitochondria, leading to distal axonal degeneration (Trevisan, 2018).

This study has examined in vivo how tweaking mitochondria fusion/fission balance perturbs mitochondria morphology and function and how these perturbations affect the distribution of mitochondria within Drosophila segmental nerves. Downregulation of Marf or Opa1 results in depletion of mitochondria at the neuromuscular junction synapse and in progressive distal loss of mitochondria along the extended axons in segmental nerves, but not in shorter photoreceptor axons. This effect was accompanied by a marked oxidative phosphorylation deficit. Simultaneous downregulation of Drp1 rescues the lethality and bioenergetic defect caused by neuronal Marf RNAi alone but ameliorates very marginally the distribution of axonal mitochondria. However, downregulation of Drp1 rescues fragmentation and aberrant distribution of axonal mitochondria induced by Opa1 RNAi without restoring viability or oxidative phosphorylation. Thus, the data show that shape is crucial for proper axonal mitochondria distribution, which in turn is largely independent of bioenergetic efficiency. However, the health of neurons depends on mitochondria function even in spite of irregular morphology and distribution. This has additional implications for mitochondria dynamics-dependent pathologies because it indicates that deficiencies observed in the absence of fusion/fission components may primarily arise from impaired mitochondrial function rather than disturbed mitochondrial transport and distribution (Trevisan, 2018).

The mitochondria fusion and fission machinery is essential for cells and genetic ablation of individual components in animal models results in death. The physiological relevance of fission/fusion processes also has become apparent from the study of patients harboring mutations in components of either fusion (Mfn2 and Opa1) or fission (Drp1) (Burte, 2015). The prime consequence of mutation or absence of fusion and fission proteins is a change in mitochondrial shape. Additionally, perturbation of mitochondrial transport and impairment of mitochondrial functions have frequently been reported. Derangement of fusion/fission dynamics affects in particular neurons with the longest axons and high energetic requirements, such as peripheral sensory and motor neurons, because their considerable length demands proper mitochondria functioning and distribution along nerves. The pleiotropic array of mitochondrial dysfunctions that follows the loss of fusion/fission players challenges an ability to explain this observation. Are long axons more vulnerable because of disrupted mitochondrial trafficking, because mitochondrial function is impaired, or both?
This work has investigated whether the spatial distribution of mitochondria along axons depends on form, function or both. This study found that in vivo in Drosophila disruption of inner or outer membrane fusion caused by Opa1 and Marf knockdown, respectively, results in mitochondria fragmentation and in a severe oxidative phosphorylation deficit. A striking change was observed in the spatial distribution pattern of axonal mitochondria in larval segmental nerves with a progressive loss of mitochondria along the axon in the distal direction. Distal loss was exacerbated in the NMJs that were essentially devoid of mitochondria. This defect was clearly length-dependent, as shorter photoreceptor axons did not display such phenotype, thus confirming that also in flies long axons are more prone to insult. The data indicate that distal mitochondria loss is unlikely to occur because of increased transport back to the neuron body for the autophagic clearance of dysfunctional mitochondria since it was found that downregulation of Opa1 or Marf heavily repressed trafficking in both the anterograde and retrograde directions. A specific role for Marf in linking mitochondria to the transport machinery through an interaction with Miro was also ruled out in flies, both because the two proteins did not coIP and because distal axonal loss is not specific for Marf but is observed after Opa1 knockdown and has been reported as a consequence of severe Drp1 mutation (Trevisan, 2018).

To directly address the importance of mitochondria morphology on axonal distribution Drp1 was downregulated in Marf or Opa1 depleted flies. It was reasoned that achieving new fusion/fission equilibrium might rescue mitochondria shape and, consequently, the Marf RNAi and Opa1 RNAi phenotypes. Surprisingly, it was found that rescue of Opa1 RNAi morphology defects by downregulation of Drp1 was associated with restoration of axonal mitochondria distribution. However, neither the bioenergetic capacity nor the lethality associated with Opa1 knockdown was rescued. In contrast, simultaneous downregulation of Marf and Drp1 led to a very robust rescue of lethality and strong restoration of oxidative phosphorylation. These double RNAi individuals, however, had fragmented mitochondrial morphology and displayed very limited rescue of axonal mitochondria distribution, suggesting that shape is more critical than function for mitochondria transport. A plausible rationalization of these results could be that Opa1-RNAi;Drp1-RNAi mitochondria are more elongated because Marf continues to fuse the outer membrane while fission is inhibited, but remain dysfunctional because disruption of inner membrane cristae organization due to the absence of Opa1 reduces their respiratory capacity. Conversely, partial recovery of a balance between outer membrane fusion and fission in Marf-RNAi,Drp1-RNAi mitochondria only marginally restores morphology and distribution but is sufficient to support normal respiratory activity and considerably ameliorate oxidative phosphorylation. This interpretation is strongly supported by electron microscopy (EM) analysis, clearly demonstrating that Opa1-RNAi;Drp1-RNAi mitochondria display severely altered internal organization while over 50% of Marf-RNAi,Drp1-RNAi mitochondria display normal organization and ultrastructure fully resembling wild-type mitochondria (Trevisan, 2018).

Available data indicate that deficiency in protein components of the mitochondrial fusion machinery reduces mitochondrial membrane potential and respiration, implying that altered mitochondrial morphology and perturbed function always go hand in hand. This study shows that the consequences of mitochondria dysmorphology and dysfunction can be dissociated, suggesting that mitochondria shape and function do not necessarily have an obligatory relationship. In fact, fragmented mitochondria with essentially normal bioenergetics capability can be generated within the nervous system of a living Drosophila. These flies contain very few mitochondria in distal axons and NMJs, but they are viable because many of their mitochondria are functional. The fact that a small number of bioenergetically active mitochondria can support normal neuronal function should not come as a surprise. Indeed, recent studies have highlighted the importance of local glycolysis to meet energy demands at synapses (Trevisan, 2018).

The genetic paradigms created have allowed separation og the independent contribution of form and function in determining the subcellular distribution of mitochondria. Proper mitochondria distribution requires quasi-normal external mitochondria morphology; however, this is not sufficient to ensure proper neuronal function, which not unexpectedly, depends on normal mitochondrial metabolic activity. One possible explanation linking mitochondria shape to their distribution is that fragmented mitochondria may not provide proper anchoring for transport adaptor molecules that reside in the outer membrane, such as Miro/Milton. Perhaps a smaller size does not warrant attachment of a sufficient number of adaptors to the individual mitochondrion for its correct mobility. This would provide a reason restoration of shape rescues the distribution of metabolically inefficient axonal mitochondria (Trevisan, 2018).

This study has generated an in vivo system in which mitochondria fragmentation is not accompanied by encumbering bioenergetic dysfunctions. Mitochondrial fragmentation is observed in a variety of pathological conditions, and the results indicate that in diseases characterized by altered mitochondrial morphology and altered axonal distribution, these defects may not be the primary cause of pathology since in the Drosophila model restoration of morphology and distribution without restoration of respiratory capacity does not permit survival (Trevisan, 2018).

A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase

Mitochondria undergo fission-fusion events that render these organelles highly dynamic in cells. A relationship exists between mitochondrial form and cell cycle control at the G(1)-S boundary. Mitochondria convert from isolated, fragmented elements into a hyperfused, giant network at G(1)-S transition. The network is electrically continuous and has greater ATP output than mitochondria at any other cell cycle stage. Depolarizing mitochondria at early G(1) to prevent these changes causes cell cycle progression into S phase to be blocked. Inducing mitochondrial hyperfusion by acute inhibition of dynamin-related protein-1 (DRP1) causes quiescent cells maintained without growth factors to begin replicating their DNA and coincides with buildup of cyclin E, the cyclin responsible for G(1)-to-S phase progression. Prolonged or untimely formation of hyperfused mitochondria, through chronic inhibition of DRP1, causes defects in mitotic chromosome alignment and S-phase entry characteristic of cyclin E overexpression. These findings suggest a hyperfused mitochondrial system with specialized properties at G(1)-S is linked to cyclin E buildup for regulation of G(1)-to-S progression (Mitra, 2009).

Mitochondrial disruption in Drosophila apoptosis

Mitochondrial disruption is a conserved aspect of apoptosis, seen in many species from mammals to nematodes. Despite significant conservation of other elements of the apoptotic pathway in Drosophila, a broad role for mitochondrial changes in apoptosis in flies remains unconfirmed. This study shows that Drosophila mitochondria become permeable in response to the expression of Reaper and Hid, endogenous regulators of developmental apoptosis. Caspase activation in the absence of Reaper and Hid is not sufficient to permeabilize mitochondria, but caspases play a role in Reaper- and Hid-induced mitochondrial changes. Reaper and Hid rapidly localize to mitochondria, resulting in changes in mitochondrial ultrastructure. The dynamin-related protein, Dynamin related protein 1 (Drp1), is important for Reaper- and DNA-damage-induced mitochondrial disruption. Significantly, it was shows that inhibition of Reaper or Hid mitochondrial localization or inhibition of Drp1 significantly inhibits apoptosis, indicating a role for mitochondrial disruption in fly apoptosis (Abdelwahid, 2007).

A role for mitochondria in apoptosis appears to be conserved from mammals to nematodes to yeast. The lack of clear evidence that mitochondria play a role in Drosophila apoptosis has prompted discussion of whether flies represent an evolutionary outlier in this highly conserved process. The data strongly suggests that mitochondrial disruption also plays a role in Drosophila apoptosis (Abdelwahid, 2007).

The data show that mitochondria rapidly become permeable to Cyt c when Rpr or Hid are expressed, both in cultured cells and in vivo. This alteration in mitochondrial permeability was also seen during DNA-damage-induced apoptosis. Importantly, it was demonstrated that the mitochondrial permeabilization during DNA-damage-induced apoptosis is dependent on the genes in the H99 interval. Taken together, these data indicate that Rpr and Hid are both necessary and sufficient for mitochondrial permeabilization (Abdelwahid, 2007).

In contrast, apoptosis induced by Actinomycin D, UV, and DIAP1 RNAi does not result in mitochondrial permeabilization. This indicates that caspase activation alone is not sufficient to induce mitochondrial permeabilization and that the mitochondrial permeabilization seen on Rpr or Hid induction is not simply a general late event in apoptosis. The efficient cell killing by Actinomycin D, UV, and DIAP1 RNAi also implies that mitochondrial permeabilization is not important for all apoptosis in Drosophila cells. Rather, it suggests that the Rpr and Hid proteins have a specific activity on the mitochondria that results in mitochondrial permeabilization to execute apoptosis in a timely manner (Abdelwahid, 2007).

The effects of Rpr and Hid on mitochondria were not limited to permeabilization. It was found that mitochondrial morphology is dramatically altered within 90 min of Rpr or Hid expression, in both S2 cells and embryos. A variety of defects were found in mitochondrial ultrastructure ranging from a rounded appearance, to bulging (and occasional rupture) of the outer mitochondrial membrane, to swelling of the matrix and disruption of the cristae. This was rarely seen with other inducers of apoptosis. Rpr and Hid may directly cause altered mitochondrial morphology or could act indirectly through other proteins localized at the mitochondria (Abdelwahid, 2007).

The absence of mitochondrial permeabilization in cells treated with DIAP1 dsRNA indicates that the mitochondrial function of Rpr and Hid is independent of their ability to inhibit DIAP1. This is confirmed by data showing that expression of DeltaN-Rpr results in mitochondrial permeabilization despite the fact that this protein lacks the necessary motif to inhibit DIAP1 antiapoptotic activity. Taken together, these data demonstrate that Rpr and Hid have dual activities in the cell, both to inhibit DIAP1 and to permeabilize mitochondria. Data from other labs have suggested that Rpr is a multifunctional protein (Thomenius, 2006). The data confirm that Rpr has multiple proapoptotic activities in the fly (Abdelwahid, 2007).

The dual functionality of Rpr and Hid parallel the recently described role of C. elegans Egl-1 in mitochondrial damage (Jagasia, 2005). Egl-1 induces apoptosis by binding to Ced-9 to promote both the activation of the caspase Ced-3 and mitochondrial fragmentation. Similarly, Rpr and Hid bind to DIAP1, displacing active caspases and act on mitochondria to promote mitochondrial disruption. One difference between C. elegans and flies appears to be the requirement for caspase activity in the mitochondrial disruption. In C. elegans, Ced-3 is not required for fragmentation but is required for apoptosis in response to fragmentation. In Drosophila, caspase activity participates in the mitochondrial changes (Abdelwahid, 2007).

Two lines of evidence support a role for mitochondrial disruption in Drosophila apoptosis. First, Rpr and Hid must localize to mitochondria to elicit a full apoptotic response. Second, if mitochondrial disruption is blocked by inhibiting Dynamin related protein 1 (Drp1) expression, a decrease is seen in apoptosis. These data clearly indicate that mitochondrial localization of Rpr and Hid is required for a full apoptotic response in S2 cells. This agrees with previous data on Rpr and also with studies on a Grim mutant lacking a mitochondrial localization signal (Claveria, 2002; Olson, 2003; Chen 2004). Mitochondrial localization of Hid has been demonstrated in a heterologous system (Haining, 1999). In the Haining study, Hid killing was not compromised in the absence of mitochondrial localization, in contrast to the current observations in Drosophila cells. A role for mitochondrial localization is also supported by the finding that two mutant forms of Hid that lack mitochondrial localization in mammalian cells behave as weak loss-of-function alleles in the fly (Abdelwahid, 2007).

The mitochondrial fission protein Drp1 is implicated in mitochondrial disruption during apoptosis in yeast, nematodes, and mammals. The current data indicate a role for this protein in Rpr-induced and DNA-damage-induced mitochondrial disruption in S2 cells and in the embryo. Furthermore, the inhibition of mitochondrial disruption after Drp1 knockdown is correlated with a decrease in apoptosis, strongly suggesting that mitochondrial disruption contributes to the apoptotic response. It is interesting to note that Drp1 plays a conserved role in apoptosis in a wide variety of organisms but seems to function downstream of different pathways. In mammals, inhibition of Drp1 blocks apoptosis in response to activation of proapoptotic Bcl-2 family members. In C. elegans, Drp1 inhibition blocks endogenous death downstream of Egl1 and Ced9, also Bcl-2 family proteins (Jagasia, 2005). Even in yeast, the role of Drp1 in cell death can be modulated by Bcl-2 family proteins. Surprisingly, in flies, Drp1 appears to be acting downstream of a different family of apoptosis inducers, the RHG proteins. It remains to be seen whether a role for the fly Bcl-2 family proteins can be established in mitochondrial disruption (Abdelwahid, 2007).

Release of apoptogenic factors, most notably Cyt c, from the mitochondria is an essential step in most apoptosis in mammalian systems. However, the current work confirms the findings of others that Cyt c, although released from mitochondria by Rpr and Hid, is not important for Rpr or Hid killing. It should be noted that Cyt c has been shown to be important in some Drosophila developmental apoptosis. In these deaths, Hid is likely to act upstream of Cyt c release. If Cyt c release is required in some cells for Hid-mediated caspase activation, why not in S2 cells? It is possible that there are both Cyt c-dependent and -independent mechanisms for activating caspases, and these may be cell-type dependent. Recent data from mice carrying a nonapoptogenic form of Cyt c supports this model (Hao, 2005), since this study suggests that there is both Cyt c-dependent and -independent apoptosis during mouse development (Abdelwahid, 2007).

If release of Cyt c is not an essential step in apoptosis in most fly cells, is another apoptosis-inducing factor released during mitochondrial disruption? In mammalian cells, release of other mitochondrial proteins such as SMAC/Diablo, Omi/HTRA2, and AIF are proposed to contribute to apoptosis (Danial, 2004). There is some evidence that released mitochondrial factors do not contribute to caspase activation in the fly. Unlike in the mammalian system, mitochondrial lysates cannot activate caspases in fly cytoplasmic lysates (Means, 2006). An alternative possibility is that mitochondrial disruption per se might contribute to apoptosis in the fly through inhibition of normal mitochondrial functions essential for cell viability. This might serve as a backup system, to maximize apoptosis in cells that express low levels of the RHG proteins. A similar role for mitochondrial disruption (Rolland, 2006) has been proposed in C. elegans (Abdelwahid, 2007).

In sum, it is concluded from these studies that Drosophila is not an outlier in evolution with regard to the involvement of mitochondria in the apoptotic process. Rather, the data indicate that mitochondrial changes contribute to Drosophila apoptosis. The findings suggest that the view of the role of mitochondria in cell death has to be broadened beyond the release of proapoptotic factors, to include the general disruption of mitochondria, ensuring that doomed cells have no chance of recovery. Such a model would fit not only the changes seen in mammalian mitochondria, but also those found in yeast, C. elegans, and flies as well (Abdelwahid, 2007).

Roles for Drp1, a dynamin-related protein, and Milton,
a Kinesin-associated protein, in mitochondrial segregation,
unfurling and elongation during Drosophila spermatogenesis

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post.meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, the FLP-FRT mitotic recombination system was used to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. It was found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. This work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation (Aldridge, 2007).

In order to define the roles for the essential genes Drp1 and milton in mitochondrial morphogenesis during Drosophila spermatogenesis, mosaic males were generated in which some spermatocytes became homozygous for mutant alleles. Homozygosity was indicated by loss of fluorescence associated with Ubi-GFP, originally expressed in heterozygous cells from the chromosome homologous to that carrying the mutation. Haploid spermatids derived from meiotic division of homozygous mutant spermatocytes were also marked by lack of fluorescence and were used to determine the post-meiotic roles of Drp1 and milton (Aldridge, 2007).

For most genes, the genotype of a pre-meiotic spermatocyte dictates the phenotype of any haploid spermatid derived thereof, regardless of which allele the spermatid inherits. Primary spermatocytes
undergo a period of extensive pre-meiotic transcription during which the mRNAs for most of the genes required for post.meiotic spermatid differentiation are transcribed. Many of these messages undergo translational repression until the times when the gene
products are needed during spermiogenesis. Post-meiotic spermatids are therefore mostly dormant transcriptionally but very active translationally. Spermatids derived from spermatocytes heterozygous for a loss of function allele typically still contain wild type mRNA and/or protein and are phenotypically normal, even if the particular spermatid
has only the mutant allele. This idea applies to the expression of Ubi-GFP as well; all spermatids, even those carrying only the CyO second chromosome, in the testes of Ubi-.GFP/CyO heterozygous males have nuclear fluorescence. Conversely, for mutant recessive alleles of genes, spermatids will show a mutant spermiogenesis phenotype only if derived from a homozygous mutant spermatocyte. This approach for assessing the roles of Drp1 and milton in spermatocytes
and spermatids is valid since homozygous mutant spermatocytes were generated simultaneously lacking Ubi-GFP and either Drp1 or Milton, and since phenotypes of the haploid spermatids derived through meiotic divisions of those cells could be subsequently characterized (Aldridge, 2007).

Since both Drp1 and milton are transcribed starting in primary spermatocytes after the gonial mitotic divisions but before meiosis, the mutant clones were generated at a stage prior to the expression of any gene product; therefore, perdurance of Drp1 or Milton in mutant clones is not a significant consideration. Furthermore, the alleles of Drp1 and milton with which were made homozygous germline clones are null or strong loss of function alleles. Drp1KG03815 is a P element insertion in the first intron of Drp1 that causes lethality when homozygous and which fails to complement other lethal Drp1 alleles. The milt92 allele contains a two base pair deletion in the coding region, and the resulting frame shift leads to a truncated Milton protein of only one third the normal length (Stowers, 2002). Phenotypes seen in milt92 germline
clones are solely due to the milt92 mutation, since homozygous milt92 FRT40A males carrying an extra wild type copy of milton are viable and fertile with normal spermatogenesis (Aldridge, 2007).

The phenotype of mutant Drp1 primary spermatocytes is consistent
with a role for Drp1 in mitochondrial fission and suggests that mitochondrial fusion and fission are normally active and counterbalanced
in primary spermatocytes, as in S. cerevisiae. After gonial mitotic divisions and before meiosis, primary spermatocytes grow dramatically in size. At an early stage during this process, mitochondria temporarily aggregate before dispersing again and multiplying. While previous studies have not indicated mitochondrial
fusion and fission during the primary spermatocyte stage, the abnormal retention of a tight cluster of mitochondria in mature primary spermatocytes lacking Drp1 is consistent with the possibility that (1) this cluster represents fused mitochondria that cannot divide; (2) mitochondria normally fuse in spermatocytes, perhaps on a constant basis, and especially during the early 'polar' spermatocyte stage when mitochondria aggregate; and (3) in wild type cells, active Drp1-mediated division is required to balance fusion and to separate the mitochondrial network into the individual
organelles seen in mature primary spermatocytes, reported to number 150 per medial cross section. While the mitochondrial fusion mediator Fzo is detectable only after meiosis, its paralog dMfn24 is likely mediating low-level mitochondrial fusion in pre-meiotic spermatocytes. In the absence of Drp1, fusion predominates and results in large mitochondrial conglomerations. The possibility that other subcellular structures may also be included in the mitochondrial clusters cannot be ruled out. The data are consistent with the abnormal clustering of mitochondria seen in the cell bodies of Drp1 mutant neurons (Aldridge, 2007).

The defect observed in Drp1 spermatocytes suggests that Drp1 is required for mitochondrial morphology at an early point in spermatogenesis. Failure of putative Drp1-mediated mitochondrial fission in Drp1 mutant primary spermatocytes, and the resulting formation of an interconnected mitochondrial mass, has serious implications for the segregation of mitochondria during subsequent meiotic divisions. In wild type testes, individual mitochondria align on the meiotic spindle during each meiotic division, enabling roughly even mitochondrial
distribution to daughter cells. If the mitochondrial material within a primary spermatocyte comprises an indivisible mass, then such segregation of mitochondria to secondary spermatocytes and then to spermatids should prove difficult, unless the force of cytokinetic division can trigger the breakage of the mitochondrial mass spread between daughter cells. However, the nature of meiotic cytokinesis makes such forced mitochondrial breakage unlikely, since cytoplasmic bridges between the meiotic products of a primary spermatocyte remain open throughout spermatid differentiation (Aldridge, 2007).

The configuration of mitochondria in early spermatids derived from homozygous Drp1 spermatocytes indeed suggested that the mitochondrial material began as an indivisible mass, which could not be divided properly during meiotic cytokinesis. The mitochondrial material in up to four cells at a time appeared connected, passing through the cytoplasmic connections between spermatids. In spermatid cysts whose cytoplasmic connections had been broken open by the pressure of the cover slip to give a syncytial appearance, the mitochondrial masses still appeared connected. Some mutant spermatids appeared to lack mitochondria, perhaps as a result of meiotic divisions in which the entire mitochondrial mass was segregated by chance to one of the other meiotic products
of the original spermatocyte. The data suggest that Drp1 has a conserved role in mitochondrial distribution during meiosis, since the Drp1 homolog Dnm1p is required for proper mitochondrial distribution during meiosis and sporulation in the budding yeast S. cerevisiae (Otsuga, 1998; Bleazard, 1999; Aldridge, 2007).

Drp1 is required not only for mitochondrial segregation during meiosis but also perhaps for mitochondrial unfurling during axoneme elongation. In wild type cells, the two mitochondrial derivatives within a Nebenkern disentangle from each other, with the large surface area of each mitochondrial derivative immediately stretched and elongated beside the growing flagellar axoneme. It is hypothesized that Drp1-mediated mitochondrial fission is required for the breaking of multiple topological links between the two mitochondrial derivatives within the Nebenkern during unfurling. Given that Drp1 cells are defective prior to and immediately after meiosis, the possibility cannot be definitively rule out that the observed unfurling defects are a secondary effect of the earlier phenomena; however, secondary effects on mitochondrial unfurling are not seen in other known mutants with early mitochondrial defects. For example, an abnormally large Nebenkern initially associated with four nuclei (resulting from a meiotic cytokinesis defect) in four wheel drive mutants properly unfurls and elongates. Furthermore, in parkin and pink1 mutants, whose Nebenkerne consist of one compartment rather than two, mitochondrial unfurling still leads to a cohesive, elongating mitochondrial derivative of largely normal morphology. In Drp1 spermatids with connected Nebenkerne, one would expect normal unfurling and elongation to lead to a maximum of eight distinct linear mitochondrial derivatives, perhaps still connected. In contrast, the mitochondrial material in Drp1
spermatids spreads out, appears massively interconnected and tangled, and does not elongate. It is therefore speculated that the lack of mitochondrial fission in Drp1 spermatids may directly interfere with mitochondrial unfurling, thereby inhibiting mitochondrial elongation (Aldridge, 2007).

In neurons, both Drp1 and Milton are required for proper transport of mitochondria to synapses. However, the basis for the defects appears to be different in each case; in Drp1 homozygous
neurons, a presumed failure of mitochondrial division leads indirectly to the unavailability of small transportable mitochondria, while in milton neurons, the defect appears to be more directly in the transport process. It was found that in spermatogenesis, the Drp1 and milton mutant phenotypes are distinct, confirming separate roles for these genes in mitochondrial morphogenesis (Aldridge, 2007).

The data are consistent with a role for Milton in some events of mitochondrial distribution in Drosophila spermatids. In spermatids derived from homozygous milt92 spermatocytes, Nebenkerne form properly, indicating that Milton is not required for mitochondrial aggregation. However, the Nebenkerne remain beside the nucleus only 36% of the time, compared to 89% in wild type spermatids. Milton thus contributes to proper anchoring of the Nebenkern in onion stage spermatids, though other gene products also must play a role in this attachment. In wild type cells at this stage, the Nebenkern resides directly beside the spot where the basal body is embedded in the nucleus. The axonemal microtubules emanating from the basal body are surrounded by a membraneous sheath, and the Nebenkern associates not with the axonemal microtubules directly but instead with cytoplasmic microtubules that also emanate from the basal body. Perhaps Milton, via an association with kinesin, helps connect the Nebenkern in stable fashion to cytoplasmic microtubules anchored to the nucleus (Aldridge, 2007).

Milton also plays a role in the elongation of mitochondria during axonemal growth. In wild type cells, when the two mitochondrial derivatives within a Nebenkern unfurl from each other, they very transiently appear as two round lobes (and are very rarely observed at this stage) but then are immediately distorted, pulled lengthwise into a leaf blade shape, presumably along the cytoplasmic microtubules. In spermatids derived from homozygous milt92 spermatocytes, the unfurled mitochondrial derivatives appear as spherical lobes for an extended period of time, suggesting that Milton normally mediates attachment of mitochondria to the cytoplasmic microtubules to enable shape changes. In wild type cells of a slightly later stage, the increasingly available surface area from the unfurling mitochondrial derivatives allows further elongation of the leaf blade structure. In contrast, the unfurling mitochondrial derivatives
in milt92 spermatids are not immediately stretched along the cytoplasmic microtubules, remaining crumpled. This early failure of elongation occurs whether or not the mitochondrial derivatives have maintained association with the nucleus. Ultimately, some mitochondrial elongation occurs in milt92 spermatids, though mitochondrial derivatives in these cells are misshapen and usually oriented improperly with respect to the nucleus. It is concluded that Milton plays an important role in mitochondrial elongation, likely through attachment to microtubules, but that other gene products mediate some mitochondrial elongation in the absence of Milton (Aldridge, 2007).

The elongation of spermatid mitochondria may involve either
(1) mitochondrial anchoring at the proximal (minus) end of cytoplasmic
microtubules and subsequent sliding of mitochondrial membranes toward the distal (plus) end, or (2) progressive immobilization
of mitochondrial membranes along growing cytoplasmic microtubules, analogous to the closing of a zipper. Milton (and perhaps kinesin) may act as they do in neurons, mediating mitochondrial movement toward the microtubule plus ends, or may serve simply to anchor mitochondria in static fashion during elongation. The decreased association of Nebenkerne with nuclei in milt92 onion stage spermatids also suggests an anchoring role for Milton at the microtubule minus end. The dynein motor protein has recently been shown to act not only as a progressive motor but also as a static anchor for cargo. Consistent with a bidirectional transport model, the non-kinesin-associated Milton isoform (Glater, 2006) and/or the testis-specific isoform (Stowers, 2002) may enable anchoring or minus-end directed movement of mitochondria toward the nucleus, while other isoforms may mediate plus.end directed mitochondrial elongation (Aldridge, 2007).

The technique udrf for generating germline clones of Drp1 and milton allowed assessment of mutant phenotypes through the mid-elongation
stages of spermatogenesis, after which point the condensed nuclei and the bundled nature of the elongating sperm (sixty four cells per cyst) made it impossible to identify individual mutant cells within a cyst. The transheterozygous male flies did not have homozygous
clones that encompassed entire cysts. Most structural defects during spermatogenesis cause sterility through failure of individualization,
which is the final investment of each sperm with its own plasma membrane and concomitant disposal of waste materials from each cell. Given the severity of the Drp1 and milton phenotypes, it is likely that individualization of mutant sperm similarly fails in these mutants. Observations that homozygous milton germline clones in a heterozygous dominant male sterile background do not confer fertility, while clones of the background chromosome do, indeed suggest that milton sperm either fail to individualize or are non-motile due to the mitochondrial defects (Aldridge, 2007).

In summary, roles have been defined for Drp1 and Milton in the specialized mitochondrial morphogenesis that takes place during spermatogenesis in Drosophila. Drp1-mediated mitochondrial division enables proper mitochondrial distribution during male meiosis as well as post-meiotic unfurling of mitochondrial derivatives (either directly or indirectly). Milton helps anchor the Nebenkern to the nucleus and subsequently mediates elongation of mitochondrial derivatives in developing spermatids. This work demonstrates that similar mechanisms for mitochondrial morphogenesis have been adapted for highly specialized use in different tissues within the organism (Aldridge, 2007).

In a forward screen for genes affecting neurotransmission in Drosophila, mutations were identified in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, evidence is provided that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. These data suggest a specific role for mitochondria in regulating synaptic strength (Verstreken, 2005).

DRP1 is the fly homolog of dynamin-related protein, a protein implicated in fission of the outer mitochondrial membrane (reviewed in Praefcke, 2004 and Rube, 2004). Mutations in Drosophila drp1 lead to dramatic defects in synaptic localization of mitochondria, but not in that of other organelles. The animals survive beyond the third instar larval stage and sometimes develop into adult flies. These and other data indicate that mitochondria in the cell bodies of the mutants are still functional. Hence, drp1 mutants allow assessment of the role of mitochondria in neurotransmission. The data show that when drp1 synapses are stimulated at high frequency, they fail to maintain normal neurotransmission. Interestingly, this defect is not due to defects in exo- or endocytosis. Rather, the data indicate that lack of synaptic mitochondria results in a specific defect in mobilizing reserve pool (RP) vesicles. Furthermore, the addition of ATP partially rescues the observed defects. Similarly, application of drugs that target mitochondrial ATP production, but not Ca2+ buffering, block RP mobilization, suggesting that mitochondrial energy production is critical for RP mobilization. Using inhibitors of myosin light chain kinase, evidence is provided that ATP production by mitochondria is limiting to myosin-propelled vesicle mobilization from the RP, downstream of PKA-mediated mobilization of RP vesicles. Hence, these data suggest a regulatory role for mitochondria in the control of synaptic strength (Verstreken, 2005).

Most studies that acutely perturb mitochondria indicate that these organelles rapidly (<5 s) buffer Ca2+ during intense stimulation. However, in drp1, in which mitochondria are largely lacking at the synapse (this work; Li, 2004), Ca2+ elevation after 30 s of 10 Hz stimulation is similar to that in controls, suggesting that mitochondria play no or a minor role in Ca2+ buffering at NMJs under the conditions tested. Instead, other Ca2+ clearance mechanisms, such as the ER or the Na+/Ca2+ pumps, may predominate early during stimulation. Interestingly, when Ca2+ uptake into the ER is blocked at the Drosophila NMJ, intense stimulation induces a steep rise in intracellular Ca2+ that quickly returns to control levels, suggesting that the ER functions as an immediate Ca2+ sink. In addition, recruitment of mitochondrial Ca2+ buffering only after prolonged stimulation supports the existence of low-affinity Ca2+. Hence, this analyses provide evidence that mitochondria are not the main determinant of Ca2+ regulation during intense stimulation at the Drosophila NMJ (Verstreken, 2005).

When mitochondrial function is acutely blocked during intense stimulation, synaptic transmission is depressed. Ultrastructural analyses in amphibian synapses have attributed this to a reduced total number of vesicles, whereas FM1-43 studies in snake terminals and sesB mutants in flies suggest impairments in vesicle cycling. Although these studies imply a function for mitochondria in neurotransmission, the role of these organelles in regulating vesicle cycling has remained elusive. These data indicate that mitochondria are central to RP vesicle cycling. (1) Neurotransmission in drp1 mutants attenuates during intense stimulation. (2) Specific FM loading protocols mostly fail to label the RP of drp1 mutants. (3) RP loading is disrupted in NMJs treated with drugs that poison mitochondrial ATP production, but not in those treated with a drug that impairs mitochondrial Ca2+ buffering. (4) Several of the functional defects described in this study are strikingly similar to those of NCAM-deficient mouse NMJs also harboring a defective reserve pool of vesicles or synapsin knockout mice lacking a reserve pool. Taken together, these observations indicate that the reserve pool vesicle cycling is disrupted in drp1 mutants (Verstreken, 2005).

A failure to label the reserve pool (RP) of drp1 mutants with FM1-43 could be caused by either a direct defect in loading or a defect in unloading of this vesicle pool, resulting in an inability to replace old unlabeled RP vesicles with new FM1-43-labeled vesicles. Although a defect in loading cannot be excluded, the data are consistent with defects in unloading and mobilization of the RP. First, when oligomycin, a blocker of mitochondrial ATP production, is added to preparations in which the RP was labeled with FM1-43, intense stimulation fails to unload the labeled RP vesicles, suggesting that mitochondrial ATP is required for their mobilization. Second, it is conceivable that a defect in RP loading results in a smaller total vesicle pool; however, the number of vesicles measured by TEM in drp1 and in controls at rest is similar. Finally, stimulation of shi;drp1 mutants at the restrictive temperature leads to the release of fewer vesicles than in controls, suggesting that drp1 mutants harbor nonreleasable RP vesicles. Interestingly, forward-filling of shi;drp1 motor neurons with ATP alleviates this defect. Hence, without excluding additional defects in the loading of RP vesicles, these data indicate that lack of synaptic ATP primarily affects the mobilization of RP vesicles (Verstreken, 2005).

It is generally assumed that mitochondria fuel many steps of the vesicle cycle, including NSF (N-ethylmaleimide-sensitive fusion protein)-mediated SNARE (soluble NSF attachment protein receptor) uncoupling and vesicle uncoating, transport, and priming, by providing ATP for these processes. However, this study provides evidence that mitochondria (when depleted by >90%) specifically limit RP mobilization and do not play a critical role in endo- or exocytosis. (1) During mild stimulation, EJPs and FM1-43 dye uptake are normal, indicating normal basal endo- and exo-cytosis. (2) After depleting the cycling vesicle pool of shi;drp1, these mutants are able to reform this pool in the same time frame as controls. (3) Although drp1 mutants exhibit rundown at intense stimulation, vesicle uptake into the ECP is normal during this paradigm. (4) drp1 boutons loaded with FM1-43 at the end of a 10 Hz train release these vesicles almost completely upon stimulation, indicating normal exocytosis. These observations demonstrate that defects in endo- and exocytosis do not account for the rundown observed during intense stimulation and suggest that synaptic mitochondrial function does not affect all steps of the vesicle cycle equally (Verstreken, 2005).

The data suggest that energy generation by mitochondria is critical for RP vesicle mobilization, as ATP partially rescues the functional defects in drp1 mutants. In addition, TPP+, a drug that affects mitochondrial Ca2+ buffering but not ATP production, shows normal mobilization of RP vesicles. In contrast, drugs that block mitochondrial ATP production show defects in RP mobilization. Finally, analyses in hippocampal synapses suggest that ECP cycling does not require much ATP, in agreement with the current observations (Verstreken, 2005).

The observation that a severe reduction in synaptic mitochondria rather specifically blocks RP mobilization suggests that this process requires most of the ATP during intense activity and that enough ATP persists in stimulated drp1 synapses to maintain exo- and endocytosis. Taking into account ATP synthesis by the few remaining mitochondria at drp1 synapses (5%-10%) and the ATP generated by glycolysis in the cytoplasm (5% of the total ATP production, it is estimated that drp1 synapses produce only 10%-15% of the ATP of control synapses. It is surmised that this is sufficient for exo-endo cycling vesicular pool cycling, but is limiting for RP mobilization (Verstreken, 2005).

The molecular mechanisms of vesicle mobilization remain, at present, poorly understood. However, an involvement of PKA and MLC has been inferred from several studies. Whereas PKA inactivation leads to increased RP vesicle tethering, active PKA results in their release. This study provides evidence that the mobilization of these untethered vesicles requires ATP produced by mitochondria, and the data further suggest that this ATP-dependent step involves myosin. The myosin complex organizes synaptic vesicle transport along actin tracts. Myosin is a major ATPase and is activated by MLCK-mediated phosphorylation of its light chain (MLC). These findings indicate that myosin uses mitochondrial-produced ATP to mobilize RP vesicles. Indeed, supplementing drp1 synapses with ATP rescues their RP mobilization defect. However, MLCK inhibitors block this effect, suggesting that activation of myosin by MLC and MLCK and the supply of mitochondrial ATP are required to mobilize RP vesicles. Recent data show that mitochondrial ATP production is regulated by synaptic activity, further highlighting the central importance of mitochondria in the regulation of synaptic strength and providing a direct link between synaptic activity and the mobilization of RP vesicles (Verstreken, 2005).

Mutations in dynamin-related protein result in gross changes in mitochondrial morphology and affect synaptic vesicle recycling at the Drosophila neuromuscular junction

Mitochondria are the primary source of ATP needed for the steps of the synaptic vesicle cycle. Dynamin-related protein (DRP) is involved in the fission of mitochondria and peroxisomes. To assess the role of mitochondria in synaptic function, a Drosophila DRP mutant combination was characterized that shows an acute temperature-sensitive paralysis. Sequencing of the mutant reveals a single amino acid change in the guanosine triphosphate hydrolysing domain (GTPase domain) of DRP. The synaptic mitochondria in these mutants are remarkably elongated, suggesting a role for DRP in mitochondrial fission in Drosophila. There is a loss of neuronal transmission at restrictive temperatures in electroretinogram (ERG) recordings. Like stress-sensitive B (sesB), a mitochondrial adenosine triphosphate (ATP) translocase mutant that was studied earlier for its effects on synaptic vesicle recycling, an allele-specific reduction in the temperature of paralysis of Drosophila synaptic vesicle recycling mutant shibire was seen in the DRP mutant background. These data, in addition to depletion of vesicles observed in electron microscopic sections of photoreceptor synapses at restrictive temperatures, suggest a block in synaptic vesicle recycling due to reduced mitochondrial function (Rikhy, 2007).

Rolland, S. and Conradt, B. (2006). The role of mitochondria in apoptosis induction in Caenorhabditis elegans: more than just innocent bystanders?. Cell Death Differ. 13: 1281-1286. PubMed ID: 16729022